Not applicable
The present invention relates generally to methods for the identification of microorganisms in liquid or liquified samples, and more particularly, but not by way of limitation, to methods using DNA for the identification of microorganisms which are indicators of contamination in liquid or liquified samples.
Most water-borne human pathogens cause infections and human disease via ingestion of fecal contaminated water or food. Various human parasites and pathogens are transmitted via human fecal contamination of water used for drinking, bathing, recreation, or washing/preparation of foods. To ensure good public health, there is a need for readily available methods to detect and enumerate pathogens in water.
While the presence of pathogens in water presents a significant public health concern, recovery of pathogens from environmental samples is generally difficult. Many fecal pathogens are infective at densities so low that water sample collection and concentration is inconvenient. Also, unpredictable physiological and morphological changes are observed in these pathogens in response to nutrient limitations and environmental stressors, and these injuries cause the organisms to exhibit atypical reactions and require specialized handling for their resuscitation. In addition, viable but unculturable organisms may be present in the water sample.
The methods commonly used to detect these pathogens were initially designed for clinical, rather than environmental, samples. However, clinical isolates are usually provided an ideal environment in which needed nutrients as well as protection from harsh environmental conditions, such as cold, heat, damaging chemicals and radiation, are readily supplied to the isolate. In contrast, environmental isolates are exposed to harsh, environmental conditions and effectively compete with organisms naturally present and adapted to life in the environment.
Pathogenic organisms are rarely readily adaptable to prolonged survival in the environment. For this reason, fecal microbial water contamination often is assessed by testing for harder and more robust, but not necessarily pathogenic, microbes, referred to as indicator organisms, such as the coliforms, especially Escherichia coli, and Enterococcus species. Indicator organisms serve to indicate whether a given water supply may be generally contaminated with fecal material without actually testing for the presence of all enteric pathogens. This contamination is viewed as predictive of the potential presence of enteric pathogens (i.e., without the presence of fecal material, the chances of these indicator organisms being present is usually remote).
Criteria for the establishment of the xe2x80x9cidealxe2x80x9d indicator include the following factors: (1) the indicator should always be present in the presence of pathogens; (2) the indicator should always be present in a predictable ratio with pathogenic organisms; (3) the indicator should be specific for fecal contamination; (4) the indicator should be able to resist water treatment and disinfection processes to the same or a slightly greater extent than the pathogens; and (5) the indicator should be detectable by simple and rapid methods.
Although coliforms have historically served as the indicator bacteria for fecal contamination in United States water supplies, the term xe2x80x9ccoliformxe2x80x9d encompasses four genera (Escherichia, Citrobacter, Enterobacter and Klebsiella) which include many species that are commonly found in the environment in the absence of fecal contamination. E. coli is the only species of coliform bacteria which is consistently and exclusively found in feces, and therefore, coliform detection methods are not specific for detecting fecal contamination in a water supply. Nonetheless, regulations based on detection and enumeration of xe2x80x9ctotal coliformsxe2x80x9d are still in effect in the United States.
Tests have also been developed to detect xe2x80x9cfecal coliformsxe2x80x9d, a subgroup of total coliforms which are thermotolerant. However, this designation is also nonspecific, as it includes not only E. coli, but also various Klebsiella strains. Despite the fact that there are substantial environmental sources of Klebsiella, and this organism is infrequently found in human feces, the use of the designation xe2x80x9cfecal coliformxe2x80x9d as well as tests to identify these organisms remain routine.
Typically, fecal coliform identification has relied on methods for distinguishing phenotype aspects such as growth or motility characteristics, and for immunological and serological characteristics. Selective growth procedures and immunological methods are the traditional methods of choice for bacterial identification, and these can be effective for the presumptive detection of a large number of species within a particular genus. However, theses methods are time consuming and are subject to error.
Selective growth methods require culturing for one to several days in selective media, followed by subjective analysis, including various metabolic, biochemical and immunochemical tests to confirm the microbiological identities of the organisms. These methods are labor, time and supply intensive, and must be performed by an experienced investigator.
Immunological detection (e.g., ELISA) is more rapid and specific, however, it still requires growth of a significant population of organisms and isolation of the relevant antigens. The sensitivity levels of currently available ELISA tests are about 104-105 organisms per milliliter, and organisms present at lower concentrations will not be detected; therefore, one or more culture steps are required in order to enrich the number of microorganisms present in the sample, and these culture steps are generally time-consuming. In addition, antigenic cross reactivity using these serological procedures can result in misidentification of organisms. Also, immunological detection typically does not distinguish living from dead cells.
Rapid processes for detecting fecal coliforms by growth in a medium containing a fluorogenic substrate (methylumbelliferyl galactoside) which is metabolized by the bacteria are described in U.S. Pat. No. 5,292,644 entitled xe2x80x9cRapid Process For Detecting Coliform Bacteria,xe2x80x9d and U.S. Pat. No. 5,518,894 entitled xe2x80x9cRapid Coliform Detection System.xe2x80x9d These processes are much less time intensive than the previously described methods, and the total amount of time required for a presence-absence result generally does not exceed seven hours.
Other methods rely on hydrolysis of methylumbelliferyl glucuronide by xcex2-glucuronidase, such as the methods described in U.S. Pat. No. 5,429,933 entitled xe2x80x9cDetection of First Generation Environmental Sourced Microbes in an Environmentally-Derived Sample.xe2x80x9d However, recent findings have shown that many coliform bacteria obtained from fecal samples do not possess xcex2-glucuronidase, which is required to metabolize this fluorogenic substrate. Further, although 97% of clinical isolates of E. coli were xcex2-glucuronidase-positive, only about 66-70% of all E. coli obtained from fecal samples were xcex2-glucuronidase-positive. Therefore, these processes would fail to detect a significant portion, about 30%, of E. coli present in the fecal samples.
Therefore, interest has turned to detection of bacterial pathogens on the basis of specific DNA sequences. Colony hybridization uses a radiolabelled unique DNA gene probe fragment isolated from chromosomal DNA of the coliform of interest to identify colonies of the organism isolated from a water sample; this technique has higher sensitivity than those described above. Methods for hybridization of oligonucleotide probes to samples for detection of an organism or a group of organisms are described in U.S. Pat. No. 5,693,469 entitled xe2x80x9cNucleic Acid Probes and Methods for Detecting Escherichia coli,xe2x80x9d U.S. Pat. No. 5,792,854 entitled xe2x80x9cDetection of Salmonella,xe2x80x9d and U.S. Pat. No. 5,795,717 entitled xe2x80x9cOligonucleotides for Detecting Bacteria and Detection Process.xe2x80x9d However, considerable time and expense is still required for hybridization methods, and a minimum of 103-104 cells per milliliter are required for detection.
Another method of detection of bacterial pathogens on the basis of nucleic acid sequence involves DNA amplification and the use of the polymerase chain reaction (PCR). Theoretically, the presence of a single microorganism can be detected by this method. U.S. Pat. No. 5,652,102 entitled xe2x80x9cAssay for Enterohemorrhagic Escherichia coli 0157:H7 by the Polymerase Chain Reactionxe2x80x9d and U.S. Pat. No. 5,298,392 entitled xe2x80x9cProcess for Detection of Water-Borne Microbial Pathogens and Indicators of Human Fecal Contamination in Water Sample and Kits Thereforxe2x80x9d both describe methods for detection of organisms by PCR and specific PCR primers for use in the methods. Patent ""102 teaches the use of primers derived from the sequences of the genes encoding the Shiga-like toxins I and II, the eaeA gene which encodes an outer membrane protein, and the 60-MDa plasmid which is present in most enterohemorrhagic E. coli (EHEC) for the detection of the specific EHEC serotype 0157. Patent ""392 teaches the use of primers derived from specific target genes for detection of groups of organisms; the target genes include the lacZ gene to detect all species from the genera Escherichia, Enterobacter, Citrobacter, and Klebsiella, the lamB gene to detect E. coli and all Salmonella and Shigella species, and the UidA gene to detect E. coli, Shigella sonnei, and Shigella flexneri. However, both of the methods described in ""102 and ""392 detect the amplification products by Southern blotting, which involves gel electrophoresis of the amplification products, transfer of the DNA to a membrane, and hybridization with a labeled primer specific for the amplification product and different from the primers used for amplification. This detection process is generally time consuming and expensive.
Thus, improved methods for detecting indicator organisms which are directly related to fecal contamination and/or the presence of pathogens which can cause waterborne disease which have reduced detection times and increased specificity and sensitivity of detection are being sought. It is to such methods, and primers used therein, that the present invention is directed.
According to the present invention, methods for the identification of bacteria in liquid or liquified samples as indicators of fecal contamination and/or the presence of pathogens which can cause waterborne disease are provided which avoid the disadvantages and defects of the prior art.
Broadly, species specific oligonucleotides based on the sequences of the E. coli specific lamB gene and the Enterococcus faecalis/faecium transposase gene Tn1546 are provided for detection and identification of these bacteria in a liquid or liquified sample.
The method of detection and identification preferably involves enrichment of the bacteria using selective culture media, recovery of bacteria from media, lysing of the bacteria to release DNA, polymerase chain reaction (PCR) amplification of the target sequences using the novel oligonucleotides, and specific detection of PCR products.
In particular, an object of the present invention is to provide novel oligonucleotides as primers for PCR assays for the specific detection and identification of Enterococcus faecalis, Enterococcus faecium and Escherichia coli. 
Another object of the present invention, while achieving the before-stated object, is to provide PCR assay methods utilizing the novel primers.
Other objects, features and advantages of the present invention will become apparent from the following detailed description when read in conjunction with the appended claims.